Project Summary: As the HIV/AIDS research field embarks on an endeavor to cure HIV-1 infection, insight into the obstacles to curing infection are essential. While most of the attention in this regard has focused on CD4+ T-cell reservoirs, far less attention has focused on whether tissue macrophages are a reservoir in HIV-1-infected individuals on suppressive antiretroviral therapy (ART). Indeed, the question of macrophage reservoirs has been polarized by recent work arguing that viral DNA in macrophages is exclusively a consequence of phagocytosis of infected CD4+ T-cells (Calantone et al., 2014). As a result, research on myeloid cell reservoirs is falling into obscurity. Infection of macrophages can only be initiated by HIV-1 variants that have the ability to use low levels of CD4 on the cell surface. We therefore propose to reveal myeloid cell reservoirs by the presence of macrophage-tropic envelopes from DNA and cell-associated RNA obtained from lymphoid tissues of ART-suppressed individuals and from the earliest recrudescing viruses obtained from individuals undergoing analytic treatment interruption (ATI). This unbiased approach does not rely on purification of myeloid cells to homogeneity nor is it obfuscated by the presence of phagocytosed CD4+ T-cells. To further validate myeloid cells as functional reservoirs, we will attempt to directly demonstrate the existence of macrophage-tropic, replication-competent virus in an accessible, macrophage-rich anatomic compartment. Specifically, we propose to: Aim 1: Identify macrophage-tropic variants in lymphoid tissue (LN, GALT) from ART-suppressed individuals and in early recrudescing variants following ATI. We will employ a semi-high throughput tropism assay, capable of identifying low-frequency, macrophage-tropic viruses to infer the existence of a macrophage reservoir. Aim 2: Establish whether alveolar macrophages support HIV-1 persistence in individuals on suppressive ART. Tropism and phylogenetic relationships between virus populations in alveolar macrophages and CD4+ T- cells from aviremic individuals will be assessed as well as isolation of macrophage-tropic virus from alveolar macrophages ex vivo These studies should, for the first time, provide definitive evidence for a viral reservoir in tissue macrophages and as such, provide the catalyst for the design of strategies to clear macrophage reservoirs and increase the likelihood of curing infection.